748 research outputs found
Sustainable Development Commission’s response to the Department for Transport on biofuels and the renewable transport fuels obligation
No full textIn this report to Department for Transport, the Sustainable Development Commission recognise that biofuels may have a part to play in reducing transport related carbon dioxide emissions. However this will depend on the appropriate safeguards being in place.Publisher PD
The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry
No full textAAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.BN/Marie-Eve Aubin-Tam LabBN/Liedewij Laan La
Structural Analysis of the Spinach Rubisco Activase AAA+ Domain by Negative Stain Electron Microscopy
No full textabstract: Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity
Tetrameric architecture of an active phenol-bound form of the AAA<sup>+</sup> transcriptional regulator DmpR
No full textThe Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.BN/Chirlmin Joo La
Avaliação em médio prazo da pressão intra-saco após correção endovascular de aneurisma de aorta abdominal com o uso de sensor sem fio
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Ciências Médicas.Justificativa: O objetivo do tratamento endovascular do aneurisma de aorta abdominal é a exclusão do saco aneurismático da circulação sistêmica. Para acompanhar a eficácia do tratamento é necessária a realização de angiotomografias seriadas. A monitorização continuada da pressão poderia diagnosticar precocemente casos em que esteja ocorrendo falha desse tratamento, auxiliando ou substituindo a tomografia. Objetivo: Avaliar a eficácia em médio prazo da medida de pressão através de sensor sem fio implantado no saco aneurismático após tratamento endovascular de aneurisma de aorta abdominal (AAA). Desenho do Estudo: Quarenta pacientes submetidos a tratamento endovascular de aneurisma de aorta abdominal receberam implante de sensor sem fio para monitorização em médio prazo da pressão intra-saco. Os dados foram analisados no primeiro, sexto, décimo segundo meses e anualmente. A cada análise, uma angiotomografia informava o diâmetro do aneurisma, presença e tipo de vazamento. A pressão arterial sistêmica assim como todos os dados pressóricos obtidos através do sensor foram coletados. Resultados: No seguimento, dos 40 sensores implantados apenas em dois o sinal não pode ser detectado. A pressão de pulso obtida apresentou boa sensibilidade em afastar a presença de vazamento quando inferior a 25mmHg no primeiro ano. Após esse período, o Índice de Endotensão mostra-se como a variável que mais se aproxima dos dados tomográficos. Na presença de vazamantos tipo I ou III, o sensor é capaz de identificar sucesso ou falha após a correção do vazamento. Até o segundo ano de seguimento, resultados apresentados pelo sensor são concordantes com dados tomográficos, a partir daí, perdem acurácia. Conclusão: Na amostra estudada não foi possível obter sensibilidade suficiente para substituir a realização da tomografia no acompanhamento dos AAA tratados por técnica endovascular. Um maior número de pacientes é necessário para estabelecer o verdadeiro papel da monitorização pressórica sem fio no seguimento do tratamento endovascular do aneurisma de aorta abdominal.Racional: The objective of endovascular treatment of an abdo-minal aortic aneurysm is to exclude the aneurysm sac from systemic circulation. In order to assess treatment effectiveness it is necessary to perform serial computed tomography (CT). Continued pressure moni-toring could provide an early diagnosis of cases in which this treatment is failing, helping or replacing CT. Objective: Evaluation of medium-term effectiveness of pressure measurement using a wireless sensor implanted in the aneurysm sac after endovascular treatment of abdominal aortic aneurysm. Method: Forty patients undergoing endovascular treatment for abdominal aortic aneurysm had a wireless sensor implanted for medium-term monitoring of intrasac pressure. The data were analyzed in the first, sixth and twelfth month and annually. At each analysis, a CT scan in-formed the aneurysm diameter, presence and risk of endoleaks. System-ic arterial pressure was collected, as well as all pressure data obtained through the sensor. Results: Of the 40 sensors implanted, the signal could not be de-tected only in two. The pulse pressure obtained presented good sensitivi-ty to rule out the presence of leakage when it was less than 25mmHg during the first year. After this period, the Endotension Index is the variable that comes closest to the CT scan data. With type I or III endo-leaks, the sensor can identify success or failure after endoleak repair. Until the second year of follow up, the results presented by the sensor agree with CT data, and then become less accurate. Conclusions: In the sample studied sufficient sensitivity could not be achieved to replace the CT when following AAA treated by the en-dovascular technique. A larger number of patients is needed to deter-mine the true role of wireless pressure monitoring when following en-dovascular treatment of abdominal aortic aneurysm
Analisis Evaluasi Penatausahaan Barang Milik Daerah (Studi Pada Pemerintah Daerah Kabupaten Pegunungan Bintang)
No full textThe objectives of this study were to evaluate the management of Government Fixed Assets in Pegunungan Bintang Regency. The analysis technique used in this study was descriptive analysis. The paper described the Analysis of Regional Property Administration Evaluation in the Pegunungan Bintang District Government. The data was shown in tables with numbers and percentages to be analyzed further with descriptive analysis. In analyzing research data the author uses qualitative descriptive analysis, also known as content analysis. The results of the study showed that the administration of regional fixed assets in terms of recording the assets, The Financial and Asset Management Agency (BPKAD) in Pegunungan Bintang Regency has carried out the bookkeeping. This comprises of process of recording regional fixed assets in lists of its users, the inventory cards, and in the database of regional’s fixed assets. Before recording the assets, at first, Asset have been identified into its classification based on the type and codes of regional fixed assets. Thus, in general, it can be concluded that the implementation of accounting in the administration of fixed assets in The Financial and Asset Management Agency of Pegunungan Bintang Regency has been well implemented. Evaluation of administration of regional fixed assets to the inventory on The Financial and Asset Management Agency in Pegunungan Bintang Regency has been carried out in accordance with mandated regulations. This can be seen from the evidence of inventory execution such as the existence of recording documents, the Database of Inventory, and documented in Inventories Books. In addition, there are reporting documents such as List of Inventory Recapitulation and List of inventory Mutations. Evaluation of administration of regional fixed assets in terms of reporting to The Financial and Asset Management Agency in Pegunungan Bintang Regency is carried out in stages based on information contained in the list of assets keepers and a list of the authority of its users and keepers. Authorities of fixed assets users have to submit assets User Reports every semester, yearly, and 5 years to assets keepers
IMPROVING CUSTOMER SATISFACTION OF SELF-SERVICE BANKING (CS DIGITAL) CASE STUDY: PT BANK AAA BRANCH X
No full textInnovation is needed in various business sectors, including banking. One of the innovations developed by banks in various countries is based on self-service banking services. Likewise, human resources (customer service-service-based) self also play an important role in customer satisfaction with a company. CS Digital is a-service based self service issued by one of the largest private banks in Indonesia. However, it was found in one branch that the use of CS Digital was not optimal. This study will measure how satisfied customers are with-based banking services, self-service namely CS Digital, compared to customer satisfaction with the quality of human (service customer service) and the author will provide some suggestions for optimizing the use of CS Digital, one of the branches of PT Bank AAA. Service quality is measured using 5 dimensions of service quality, convenience, reliability and security, responsiveness, personalization, and tangible and customer satisfaction indicators. From these attributes, it will be calculated how big the gap is between customer expectations and company performance. The calculation of the gap is assisted by the method Importance Performance Analysis (IPA). In this study, there are several problems related to the operation of CS Digital that need to be resolved. These problems include ease of use of CS Digital, lack of clear directions for new users, slow service, and CS Digital is not the first choice for customers to make transactions. Fishbone is used to analyze the root of the problem and to find out the cause and effect of problems that occur at PT Bank AAA branch X. In addition to the fishbone method, the author also uses theories regarding service quality to find the main problems in PT Bank AAA branch X. The root of the problem from PT Bank AAA branch X, I tried to give suggestions to optimize the use of CS Digital so that it can help reduce queues and increase branch productivity as expected.Keywords: Customer satisfaction, service quality, fishbon
IMPROVING CUSTOMER SATISFACTION OF SELF-SERVICE BANKING (CS DIGITAL) CASE STUDY: PT BANK AAA BRANCH X
No full textInnovation is needed in various business sectors, including banking. One of the innovations developed by banks in various countries is based on self-service banking services. Likewise, human resources (customer service-service-based) self also play an important role in customer satisfaction with a company. CS Digital is a-service based self service issued by one of the largest private banks in Indonesia. However, it was found in one branch that the use of CS Digital was not optimal. This study will measure how satisfied customers are with-based banking services, self-service namely CS Digital, compared to customer satisfaction with the quality of human (service customer service) and the author will provide some suggestions for optimizing the use of CS Digital, one of the branches of PT Bank AAA. Service quality is measured using 5 dimensions of service quality, convenience, reliability and security, responsiveness, personalization, and tangible and customer satisfaction indicators. From these attributes, it will be calculated how big the gap is between customer expectations and company performance. The calculation of the gap is assisted by the method Importance Performance Analysis (IPA). In this study, there are several problems related to the operation of CS Digital that need to be resolved. These problems include ease of use of CS Digital, lack of clear directions for new users, slow service, and CS Digital is not the first choice for customers to make transactions. Fishbone is used to analyze the root of the problem and to find out the cause and effect of problems that occur at PT Bank AAA branch X. In addition to the fishbone method, the author also uses theories regarding service quality to find the main problems in PT Bank AAA branch X. The root of the problem from PT Bank AAA branch X, I tried to give suggestions to optimize the use of CS Digital so that it can help reduce queues and increase branch productivity as expected.Keywords: Customer satisfaction, service quality, fishbon
The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain
No full textThe plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated
Limited Proteolysis of the AAA+ Protein Rubisco Activase from Nicotiana tabacum
No full textabstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is widely accepted as the world's most abundant enzyme and represents the primary entry point for inorganic carbon into the biosphere. Rubisco's slow carboxylation rate of ribulose-1,5-bisphosphate (RuBP) and its susceptibility to inhibition has led some to term it the "bottle neck" of photosynthesis. In order to ensure that Rubisco remains uninhibited, plants require the catalytic chaperone Rubisco activase. Activase is a member of the AAA+ superfamily, ATPases associated with various cellular activities, and uses ATP hydrolysis as the driving force behind a conformational movement that returns activity to inhibited Rubisco active sites. A high resolution activase structure will be an essential tool for examining Rubisco/activase interactions as well as understanding the activase self-association phenomenon. Rubisco activase has long eluded crystallization, likely due to its infamous self-association (polydispersity). Therefore, a limited proteolysis approach was taken to identify soluble activase subdomains as potential crystallization targets. This process involves using proteolytic enzymes to cleave a protein into a few pieces and has previously proven successful in identifying crystallizable protein fragments. Limited proteolysis, utilizing two different proteolytic enzymes (alpha-chymotrypsin and trypsin), identified two tobacco activase products. The fragments that were identified appear to represent most of what is considered to be the AAA+ C-terminal all alpha-domain and some of the AAA+ N-terminal alpha beta alpha-domain. Identified fragments were cloned using the pET151/dTOPO. The project then moved towards cloning and recombinant protein expression in E. coli. NtAbeta(248-383) and NtAbeta(253-354) were successfully cloned, expressed, purified, and characterized through various biophysical techniques. A thermofluor assay of NtAbeta(248-383) revealed a melting temperature of about 30°C, indicating lower thermal stability compared with full-length activase at 43°C. Size exclusion chromatography suggested that NtAbeta(248-383) is monomeric. Circular dichroism was used to identify the secondary structure; a plurality of alpha-helices. NtAbeta(248-383) and NtAbeta(253-354) were subjected to crystallization trials.Dissertation/ThesisM.S. Biochemistry 201
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